REAGENT STRIPS

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REAGENT STRIPS

• Storage
• Protect from moisture and excessive heat.
• Store at room temp. Do not refrigerate.
• Keep container tightly capped.
• Do not remove desiccant from container.
• Do not use beyond expiration date.
• Handling
• Review manufacturers instructions with each new
  lot number
• Remove strips from bottle for immediate use.
  Recap.
• Check for discoloration.
• Keep away from bleach, acids, fumes, etc.

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REAGENT STRIPS (continued)

• Testing
• within 30 minutes to 2 hours
• fresh, well-mixed, unspun
• at room temperature
• Do not touch test pad area
• Dip briefly, but completely - app. 1 second
• Drain off excess urine, avoid runover
• Do not lay strip on bench
• Compare test areas to color chart on bottle
• Read at specified times
• Know sources of error, interfering substances,
  sensitivity, and specificity for each strip.
• When automated, follow instruments operating
  manual.

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Reporting

• Standard terms must be used
• Quantitative
• Concentration mg/dL
• Plus system
• Qualitative
• Small, moderate, large
• Negative/positive/WRR (normal)

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pH

• The strip contains the indicators methyl red and
  bromthymol blue. The give colors over the pH
  range of 5-9. Colors range from orange through
  yellow and green to blue.
• Reference value on normal diet - 4.6 to 8.0

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CLINICAL SIGNIFICANCE pH

• Acid pH lt6.0
• Diet (high protein, meat, cranberries)
• Acidifying drugs to prevent alk. stone formation
• Abnormal crystalluria (bilirubin, cystine,
  tyrosine, leucine, cholesterol )
• Uric acid stone formers
• Acidosis and uncontrolled Diabetes mellitus
• Hypokalemia
• Starvation

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pH (continued)

• Alkaline pH gt6.5
• Diet (vegetarian and citrus fruits)
• Alkaline tide produced after a meal
• Metabolic and respiratory alkalosis
• Renal tubular diseases (Fanconis syndrome)
• Alkalizing drugs to treat acid calculi formation
• Genitourinary tract infections
• pH gt8.0
• Contamination or old urine (not suitable for
  testing)

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Protein

• Reagent strip testing is based on the principle
  of protein error of the indicators. Protein in
  the form of albumin accepts ions from the
  indicator, which is buffered in a very acid
  medium. Color changes from shades of green to
  blue.
• Most significant for early detection of renal
  disease.

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CLINICAL SIGNIFICANCE PROTEIN

• Reference value
• Random urine negative (not detectable) lt10mg/dL)
• 24 Hour lt150mg or 10 mg/dL
• Composition
• Albumin, 1/3a
• Globulins, 2/3
• Tamm-Horsfall mucoprotein (25)
• Pathology gt30mg/dL or on daily output
• Heavy gt3g/day
• Moderate gt1-3 g/day
• Minimal lt1g/day

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CLINICAL SIGNIFICANCE PROTEIN (CONTINUED)

• Benign
• Functional
• exercise, fever, stress, exposure to cold
• Orthostatic
• positional, renal congestion
• Pregnancy
• transient, investigate cause
• Pathologic
• Prerenal overflow of low mole weight proteins
• IgG light chains (Bence Jones proteins)
• acute phase
• hemoglobin, myoglobin

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CLINICAL SIGNIFICANCE PROTEIN (CONTINUED)

• Pathologic
• Renal Glomerular Pattern
• Group A strep and SLE glomerulonephritis
• Hypertensive and diabetic nephropathy
• Nephrotic syndrome, tumors, infections, toxic
  agents
• Renal Tubular Pattern
• Acute and chronic pyelonephritis
• Interstitial nephritis
• Renal tubular acidosis, rejection of kidney
  transplant
• Post Renal
• Inflammation/infection bladder, renal pelvis,
  ureter, prostate, external genitalia

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SSA Testing for Protein

• When mixed with weak sulfosalicylic acid (SSA),
  all urine proteins will denature and precipitate
  at room temperature. The degree of which is
  graded and reported in semi-quantitative terms.
• Albumin, globulins, glycoproteins, and
  Bence-Jones protein are detected.
• False positive caused by x-ray contrast media,
  penicillin, sulfonamides, tolbutamides
• False negative caused by highly alkaline urine

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Microalbuminuria

• Protein (albumin) that cannot be detected by
  routine dipstick for protein
• Sensitive methods needed to detect 10-20mg/L or
  1-2 mg/dL
• Immunochemical
• Dye binding
• Clinical significance
• Early management of kidney disease in diabetes,
  hypertension, or peripheral vascular disease

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Glucose

• The dipstick determination of glucose is based on
  a double sequential enzymatic reaction using the
  specific glucose oxidase/peroxidase reaction in
  the presence of glucose and a chromogen.
• The Clinitest or Benedicts Reaction is based on
  the ability of reducing substances to reduce
  copper sulfate to cuprous oxide in the presence
  of a chromogen, which changes color from blue to
  orange. Test is performed to screen for
  non-glucose reducing sugars in infants and
  children under 2 years old.

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Clinical Significance of Glucose

• Reference value
• No detectable amount present in urine by dipstick
  method (lt50 mg/dl)
• Positive values found when renal threshold for
  glucose is exceeded (160-180 mg/dl)
• Diabetes mellitus (DM)
• Impaired tubular reabsorption
• Pregnancy with latent DM

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COMPARISON of REAGENT STRIP vs. CLINITEST

• Strip Clinitest Cause
• Positive Negative Sensitivity of methods
• Oxidizing contaminants/bleach
• Deteriorated Clinitest tablets
• Negative Positive Non-glucose reducing
  substance
• Deteriorated reagent strips
• Reagent strip interferences Ascorbic
  acid (vitamin C)

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Ketones

• Conditions that result in increased and or
  incomplete fat metabolism can produce metabolic
  intermediary fat products in the urine and blood.
  The three ketone bodies present in urine are
  acetoacetic acid (20), acetone (2), and
  betahydroxybutyric acid (78).
• Acetoacetic acid and acetone react with
  nitroprusside in an alkaline medium to form a
  violet dye complex. Basis of dipstick.
• Betahydroxybutyric acid is not detected with
  dipstick.

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Clinical Significance Acetone

• No detectable ketones present in normal urines.
• Positive values
• Diabetic ketosis (ketonuria)
• Loss of carbohydrates due to fever, vomiting,
  weight loss, starvation, diarrhea, stress
• Lactic acidosis caused by liver/renal failure,
  salicylate overdose
• Interfering factors
• False positive pigmented urines
• False negative delay in testing

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Nitrite

• This test depends on the conversion of nitrate to
  nitrite by the action on Gram negative bacteria
  that contain reductase enzymes in the urine.
• At the acid pH of the reagent area, nitrite in
  the urine reacts with an aromatic amine to form a
  diazonium salt, followed by a coupling reaction
  with benzoquinoline to produce a pink color.
• Three factors must be present
• Reductase producing bacteria must be present
• Urine must be retained in bladder long enough to
  convert nitrates to nitrites (4 hours)
• Nitrates must be present

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CLINICAL SIGNIFICANCE NITRITE

• Reference value
• No nitrites present
• Urine must be FRESH
• Improper storage will result in false positive
• Positive nitrites
• Screen symptomatic and asymptomatic UTI
• Common infecting organisms
• Enterobacter, Citrobacter, Escherichia, Proteus,
  Klebsiella, Pseudomonas
• Definitive diagnosis made by urine culture
• Non-reductase producing microorganisms will be
  negative for nitrites

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Leukocyte Esterase

• Granulocytic leukocytes contain esterase activity
  that catalyze the hydrolysis of an amino acid
  ester to form an aromatic compound which reacts
  with a diazonium salt to produce a color change
  from beige to purple on the dipstick pad.
• All positive reactions require a microscopic exam
  of the sediment.

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CLINICAL SIGNIFICANCE LEUKOCYTE ESTERASE

• Reference value
• 0-5 white cells/hpf
• females 0-8 WBC/hpf or app. 10 WBC/uL (vaginal
  discharge can cause false positive)
• Screens for urinary tract inflammation
• kidney (pyelonephritis)
• bladder (cystitis)
• urethra (urethritis)
• Leukocyturia can occur with or without bacteria

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LEUKOCYTE-NITRITE

• Combination on FRESH urine is
• Cost effective tool to screen for UTI
• Provides 97 predictive value for negative
  culture when both tests are negative
• Improved care in asymptomatic patient

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Blood

• Dipstick will detect blood by sensing heme that
  is present in red cell, hemoglobin, and
  myoglobin. Based on the pseudoperoxidase activity
  of heme in the presence of an organic peroxide
  and a benzidine chromogen.
• Hematuria in tact red cells present in urine
  (scattered green dots)
• Hemoglobinuria presence of hemoglobin from lysed
  red cell in urine (diffuse green color)
• Myoglobinuria presence of heme protein from
  muscles in urine (diffuse green color)

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CLINICAL SIGNIFICANCE BLOOD

• Reference value
• 0-5 erythrocytes/mL or 0-2 RBC/hpf
• Hematuria - intact red cells
• renal disease, calculi, tumors, infections
• bleeding in kidneys or lower urinary tract
• Hemoglobinuria - free hemoglobin
• intravascular hemolysis as seen in incompatible
  blood transfusions, AIHA, G6PD, etc.
• Myoglobinuria - heme muscle protein
• acute destruction of muscle fibers
  (rhabdomyolysis)
• crush/trauma injuries
• Excessive exercise can cause all above

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COMPARISON of URINE FINDINGS

• Findings Hematuria Hemoglobinuria
  Myoglobinuria
• Color pink, red, smoke clear pink, red,
  brown clear red, brown
• UA Blood strip positive dots positive
  diffuse positive diffuse
• UA Protein strip Renal 4 pos/neg pos/neg
• Nonrenal /-
• UA RBCs many occasional occasional
• UA Casts Renal RBC Hemoglobin Myoglobin Nonre
  nal none

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Bilirubin

• The heme released from red cells is converted to
  the yellow bile pigment biliribin by a series of
  complex reactions in liver. A small amount is
  excreted under normal circumstances and is not
  detected in the urine with the dipstick.
• When present, the Diazo Reaction is based on the
  coupling of bilirubin with a diazonium salt in an
  acid medium to form a colored azo-dye complex.

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Ictotest for Bilirubin

• Highly pigmented urines can cause false positive
  reactions. Confirmation is required by testing
  with the Ictotest tablet test for bilirubin.
• This diazo tablet method is very sensitive to low
  levels of bilirubin.
• Pigments will be removed by the absorbent pad
  supplied with the test.

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CLINICAL SIGNIFICANCE BILIRUBIN

• Reference value
• not detected with reagent strips lt0.02 mg/dL
• Positive findings
• obstruction to bile flow from liver
• gallstones and neoplasms of pancreas
• inflammation and swelling of liver cells
• acute viral hepatitis, drug indued cholestatsis
• acute alcoholic hepatits/cirrhosis
• congenital hyperbilirubinemias
• Dublin-Johnson and Rotor

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Urobilinogen

• Collectively referred to as the end products of
  bilirubin metabolism. Colorless reduction product
  of bilirubin which is oxidized by normal
  intestinal bacteria to brown pigment that is
  excreted in the feces.
• Based on the Ehrlich Reaction in an acid medium
  to form a red color.

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CLINICAL SIGNIFICANCE UROBILINOGEN

• Reference value
• up to 1 mg/dL or 1 Ehrlich Unit
• greater in PM (alkaline tide after meals)
• up to 2 mg/dL transition from normal to abnormal
• decrease or absence cannot be determined with
  strip
• Increased values
• liver damage viral hepatitis, cirrhosis, drugs,
  toxins
• infections of biliary tree (cholangitis)
• hemolytic anemias and intravascular hemolysis
• increased enteric production
• Absent
• obstruction of bile duct
• absence of intestinal flora

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UA BILIRUBIN UROBILINOGEN in UNCOMPLICATED
JAUNDICE

• Condition Bilirubin Urobilinogen
• Normal Negative up to 2 EU/dL
• Hepatic Disease Positive Increased (/-)
• Obstructive Disease Positive (/-) Absent
• Hemolytic Disease Negative Increased

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QUALITY ASSURANCE

• Facilities and Resources
• OSHA compliance
• Proficiency Testing
• Personnel
• Qualifications, education and training,
  competency
• Review
• Procedure Manual
• NCCLS GP2-A2
• Controls, Standards, Reagents
• Equipment and Instruments
• Reporting of Results

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COMPETENCY ALERTS REAGENT STRIP TESTING

• Directly Observe
• Followed SOP and manufacturers instruction
• labeled date received, opened, expired
• Removed strip immediately before test run
• Replaced cap
• Performed test on well mixed, unspun urine
• Performed daily maintenance/function checks
• Interpretation of color changes for strips or
  tablets
• Performed confirmatory testing as indicated
• Performed Clinitest on nursery or pediatric
  urines
• Followed SOP step-by-step

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COMPETENCY ALERTS (continued)

• Monitor and Review
• Compliance with QC as defined in SOP
• Results logged on scheduled frequency of use
• Parallel testing
• PM and service logs signed
• Critical values reported on interim worksheets
  WHO, WHAT, WHEN.
• Standard units of measure defined in SOP are used
  to report qualitative and quantitative results.

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COMPETENCY ALERTS (continued)

• Assessment of Test Performance
• Proficiency testing
• Internal blind samples of known chemical
  concentration
• Problem Solving
• Resolve discrepant results
• Investigate and resolve delta checked results
• Specimen referred for definitive testing (UA
  culture) based on reagent strip results. Policy
  defined in SOP.
• Resolution of out-of-control results for known
  reference controls.

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MICROSCOPIC EXAM of URINARY SEDIMENT

• CLIA88 Complexity
• Moderate
• Provider Performed Microscopy (PPM)
• physician, midlevel practitioner, or dentist
• brightfield or phase microscopy
• Specimen of Choice
• Fresh first morning, midstream, clean catch
• Examine within 2 hours
• Specific gravity gt1.010
• pH acid

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IDENTIFIABLE SEDIMENT ENTITIES

• Hematopoietic cells
• Red blood cells (RBC)
• dysmorphic
• white blood cells (WBC)
• glitter
• eosinophils
• lymphocytes , histiocytes and macrophages
• Epithelial cells
• Transitional (urothelial)
• Squamous
• Renal tubular
• oval fat bodies

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• Casts
• Matrix Hyaline and Waxy
• Cellular RBC, WBC, Epithelial, Mixed, Bacteria
• Inclusion Granular, Fatty, Hemosiderin, Crystal
• Pigment Hemoglobin, Myoglobin, Bilirubin
• Size Broad or Wide
• Crystals
• Microorganisms
• bacteria, yeasts, parasites
• Miscellaneous
• spermatozoa, mucus, artifacts and contaminants